Removal of lipopolysaccharides from protein-lipopolysaccharide complexes by non flammable solvents.

ABSTRACT

During the production of recombinant proteins from gram negative bacteria, lipopolysaccharides (LPS, endotoxin) are released along with the protein of interest. In many instances, LPS will copurify with the target protein due to specific or non-specific protein-ILPS interactions. We have investigated the ability of alkanediols to effect the separation of LPS from protein-LPS complexes while the complexes are immobilized on anion or cation exchange chromatographic media. Alkanediols provide a safer alternative to the use of other organics such as alcohols or acetonitrile due to their lower toxicity and decreased flammability. In addition, they are less costly than many of the detergents that have been used for such purposes. LPS removal efficiency increased with increasing alkane chain length. 1,2-alkanediols were more effective than terminal alkanediols in the separation of LPS from protein LPS complexes.

FIELD OF THE INVENTION

Embodiments of the present invention generally relate to the removal oflipopolysaccharides (LPS) from protein-lipopolysaccharide complexes.

BACKGROUND OF THE INVENTION

Lipopolysaccharide (LPS) is a major component of the outer membrane ofgram negative bacteria. The endotoxic component of LPS is the lipid Aportion. It comprises 1,6-linked D-glucosamine residues that aresubstituted with up to six acyl chains and a core polysaccharidestructure to which additional polysaccharide repeating units may beattached. Endotoxin is a potent activator of the innate immune system atlow doses while at higher doses endotoxin induces a number of otherphysical reactions including septic shock and death (Heine et al.,2001). Contamination of therapeutic products with endotoxins istherefore a primary concern for the manufacturers of such products.

Many recombinant proteins are produced in the gram negative bacteriaEscherichia coli. The removal of LPS from these recombinant proteins canbe a complicated but essential process especially if the proteins aredestined for therapeutic uses. Many different processes have beendeveloped for the removal of LPS from proteins based on the uniquemolecular properties of the endotoxin molecules. These include LPSaffinity resins, two-phase extractions, ultrafiltration, hydrophobicinteraction chromatography, ion exchange chromatography, and membraneadsorbers (reviewed by Petsch and Anspach, 2000). These procedures havevarying degrees of success in the separation of LPS from proteins, whichin a large part is dependent on the properties of the protein ofinterest.

Often, during the production of recombinant proteins, difficulties inthe separation of LPS from proteins are encountered due to protein-LPSinteractions. For example, and not by way of limitation, in recombinantproteins produced from an E. Coli expression system. Several of thepublished procedures for the separation of LPS from proteins have beeninvestigated, including, but not necessarily limited to, denaturinghydrophobic interaction chromatography (Wilson et al., 2001) and the useof ethanol, isopropanol (Franken et al., 2000), or detergent (Fiske etal., 2001) washes while the protein was immobilized on ion-exchangechromatographic media.

Some experiments have shown that alcohol and detergent washes during ionexchange chromatography are effective in reducing the protein associatedLPS levels while poor separation of LPS from the proteins was obtainedby the denaturing HIC procedure. The detergents (Zwittergent 3-12 or3-14) were shown to be more effective washing agents than the alcohols.Improved LPS clearance was also been achieved while the LPS-proteincomplexes were bound to a cation exchange resin as opposed to an anionexchange resin but the washing procedures used to remove LPS wereeffective on both matrices. When the wash procedure is performed on acation exchanger, once the LPS-protein interactions have been disruptedthe LPS should be washed out of the column while the protein isretained. During anion exchange chromatography, the LPS, beingnegatively charged at most pHs, remains bound to the resin along withthe protein. Even though the alcohol and detergent washes weresuccessful at reducing the levels of LPS in the LPS-protein complexes,scaling up and implementing any of these procedures in a manufacturingsetting would not be practical. The concentrations of ethanol andisopropanol required to effectively reduce the LPS levels of the LPSbinding proteins were greater than 50% (v/v). At these concentrations,these solutions are considered flammable liquids and as such impose manysafety and operational restrictions.

The detergents, even though very effective at reducing LPS levels, arerelatively expensive and would add significant cost to a manufacturingprocess and may affect the bioactivity of the protein of interest.Accordingly, Alternative chemicals are desired that could safely andcost effectively be used in place of the alcohols or detergents aswashing agents for the separation of LPS from proteins duringchromatographic unit operations. Ideally, these chemicals would berelatively inexpensive, well defined chemically, present minimal safetyissues, and have minimal impact on the bioactivity of the protein inquestion when implemented into a process.

SUMMARY OF THE INVENTION

Embodiments of the present invention generally relate to the ability ofalkanediols to separate lipopolysaccharides (LPS) or other endotoxinsfrom proteins. In an embodiment, alkanediols are able to effect theseparation of LPS from LPS-protein complexes. Accordingly, embodimentsof the present invention generally relate to processes using alkanediolsto effect separation of LPS from LPS-protein complexes. In furtherembodiments, the complexes are immobilized on a resin and the LPSseparated therefrom.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a is an illustration of the SP Sepharose Fast Flow ElutionProfile of BODIPY-LPS. The dotted line is UV signal at 280 nm and thesolid line corresponds to BODIPY fluorescence expressed in relativefluorescence units (RFU).

FIG. 1 b is an illustration of the SP Sepharose Fast Flow ElutionProfile of BODIPY-LPS-Transferrin Complex. The dotted line is UV signalat 280 nm and the solid line corresponds to BODIPY fluorescenceexpressed in relative fluorescence units (RFU).

FIG. 2 is an illustration of BODIPY-LPS Elution Profiles ofBODIPY-LPS-Transferrin Complexes on SP Sepharose Fast Flow inConjunction with Alkanediol Washes. BODIPY-LPS-transferrin complexeswere generated, loaded onto a SP Sepharose Fast Flow column and thecolumn washed with 50% solutions of 1,4-butanediol, 1,6-hexanediol, or1,2-hexanediol.

FIG. 3 is an illustration of the reduction of BODIPY-LPS fromBODIPY-LPS-Transferrin Complexes in SP Sepharose Fast Flow Eluates byAlkanediols. Zero percent reduction corresponds to a control run withoutan alkanediol wash. 1, 1,2-hexanediol; 2, 1% Zwittergent 3-14; 3,1,6-hexanediol; 4, ethylene glycol; and 5, 1,4-butanediol.

FIG. 4 is an illustration of the reduction of BODIPY-LPS fromBODIPY-LPS-BSA complexes in SP Sepharose Fast Flow eluates byAlkanediols. Zero percent reduction corresponds to a control run withoutan alkanediol wash. 1, 1,2-hexanediol; 2, 1% Zwittergent 3-14; 3,1,2-butanediol; 4, 1,6-hexanediol; 5, 50% isopropanol; 6, 75% ethanol;7, 1,4-butanediol; and 8, ethylene glycol.

FIG. 5 is an illustration of viscosities of Alkanediol, Isopropanol,Ethanol, and Zwittergent Solutions in 100 mM Acetate, pH 4.5. Allsolutions were prepared with 100 mM Acteate buffer, pH 4.5. 1, 50%1,6-hexanediol; 2, 50% 1,2-hexanediol; 3, 50% 1,4-butanediol; 4, 50%1,2-butanediol; 5, 50% Ethylene glycol; 6, 50% Isopropanol; 7, 75%Ethanol; 8, 1% Zw 3-14; and 9, 100 mM Acetate, pH 4.5.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “alkanediol” means and refers to a non-aromaticsaturated hydrocarbon with the general formula C_(n)H_(2n)(OH)₂.

As used herein, the terms protein-LPS complex and LPS-protein complexshall be synonymous. The protein-LPS complex may be a loose association,such as, for example and not by way of limitation, by intramolecularforces/intermolecular forces, or binding.

Generally, embodiments of the present invention relate to processes forseparating lipopolysaccharides or endotoxins fromprotein-lipopolysaccharide (or other endotoxin comprising substance)complexes. In an embodiment, a process for separatinglipopolysaccharides from protein-lipopolysaccharide complexes comprisesthe step of washing a protein-LPS complex with an alkanediol containgsolution whereby at least a portion of the LPS is removed and/orseparated from the protein.

In an embodiment, the LPS-protein complex (or other endotoxin complex)is produced and/or formed during the production of proteins, such as,but not limited to recombinant proteins.

Embodiments of the present invention may use any protein. Variousexamples include, but are not limited to, Bovine albumin (BSA), bovineholo-transferrin, lactoferrin from bovine milk, lysozyme from chickenegg whites, heat shock proteins, heat shock fusion proteins and thelike. The various proteins may be simple or complex, small or large.

Various processes and/or expression systems may be used for theproduction of recombinant proteins. Any expression system may be usedfor the production of recombinant proteins. In an embodiment, theexpression system is bacterial. In such embodiments, gram negativebacterial expression systems may be used, such as an E. coli expressionsystem. However, other types of bacterial systems may also be used, forexample, and not by way of limitation, Caulobacter crescent and Proteusmirabilis.

In general, any system comprising endotoxin(s) can use embodiments ofthe present invention to separate the endotoxin from the protein. Forexample, in an embodiment endotoxin(s) are an impurity in a systemcomprising proteins. It is well known in the art that endotoxin(s) can,in some instances, be found as contaminants in raw materials or duringprocessing. Such endotoxins can be removed and/or separated by variousembodiments of the present invention.

Any alkanediol may be used with various embodiments of the presentinvention. Suitable, non-limiting examples include, 1,5-pentanediol,1,6-hexanediol, 1,2-hexanediol, 1,2-butanediol, 1,4-butanediol, and1,7-heptanediol. In an embodiment, alkanediols of the present inventionare long chain alkanediols. Alkanediols provide increased safety overthe commonly used eluents, like acetonitrile, ethanol, and methanol,since the alkanediols are all nonflammable compounds. Further,alkanediols are soluble in water and they are not cost prohibitive for alarge number of processes.

In other embodiments, various processes are utilized to assist and/orfacilitate separation of the protein-LPS complex. In various examples ofthese embodiments, the protein-LPS complex is attached to a substrate.Various methods of attachment include retaining, attracting, binding,applying, immobilizing and/or removably affixing to a substrate. Eitherthe protein or the LPS may be attached. In an embodiment, theLPS-protein complex is bound or immobilized on a resin. Suitable resintypes include, but are not limited to affinity resins, anion exchangeresins, cation exchange resins, and the like, such as a SP SepharoseFast Flow resin (SPSFF resin). However, the choice of resins is a matterof routine skill in the art and can be made to serve the particularneeds of the process.

In an embodiment, the LPS-protein complex is bound to an ion exchangeresin under conditions such that the protein binds to the resin of thecolumn. An alkanediol wash solution is then applied to the columnwhereby at least a portion of the LPS is separated from the LPS-proteincomplex and elutes with or about the alkanediol. In an embodiment,greater than 50% of the LPS is separated from the LPS-protein complex.In an alternate embodiment, greater than 75% of the LPS is separatedfrom the LPS-protein complex. In another embodiment, greater than 80% ofthe LPS is separated from the LPS-protein complex. In yet anotherembodiment, greater than 85% of the LPS is separated from theLPS-protein complex. In an alternate embodiment, greater than 90% of theLPS is separated from the LPS-protein complex. In another embodiment,greater than 95% of the LPS is separated from the LPS-protein complex.In another embodiment, greater than 97% of the LPS is separated from theLPS-protein complex. In another embodiment, greater than 99% of the LPSis separated from the LPS-protein complex. In another embodiment,greater than 99.9% of the LPS is separated from the LPS-protein complex.

In an embodiment, after separation and/or removal of the LPS, theprotein can be eluted from the resin. The protein may eluted by meanscommon in the art and as appropriate for the resin. In an example, forion exchangers changes in pH and/or increased conductivity can be usedto elute the protein.

In an embodiment, 50% of the protein eluted from the resin is free ofLPS or other endotoxin. In an alternate embodiment, 75% of the proteineluted from the resin is free of LPS or other endotoxin. In an alternateembodiment, 80% of the protein eluted from the resin is free of LPS orother endotoxin. In an alternate embodiment, 85% of the protein elutedfrom the resin is free of LPS or other endotoxin. In an alternateembodiment, 90% of the protein eluted from the resin is free of LPS orother endotoxin. In an alternate embodiment, 95% of the protein elutedfrom the resin is free of LPS or other endotoxin. In an alternateembodiment, 97% of the protein eluted from the resin is free of LPS orother endotoxin. In an alternate embodiment, 99% of the protein elutedfrom the resin is free of LPS or other endotoxin. In an alternateembodiment, 99.9% of the protein eluted from the resin is free of LPS orother endotoxin.

In various embodiments, another wash is utilized to remove/separate theprotein from the resin. In an alternate embodiment, the protein eluteswithout a wash, such that the resin only slowed the protein elution,thereby separating the LPS from the protein. The degree of separationcan be varied depending upon the resin used. Likewise, a resin can beconstructed to change the elution profile of the LPS-protein complexsuch that the LPS or protein component elutes at a different time thanthe other component.

Accordingly, various embodiments of the present invention compriseprocesses such as:

A process for removing an endotoxin from recombinantly produced proteinscomprising a Lipopolysaccharide (LPS)-protein complex comprising thesteps of:

immobilizing the complex to an ion exchange resin;

washing the resin with an alkanediol whereby at least a portion of theLPS is separated from the complex; and,

eluting at least a portion of the protein from the resin.

In alternate embodiments, the LPS is affixed to the resin and theprotein is first eluted.

In further embodiments, the present invention comprises a process fordisrupting a lipopolysaccharide (LPS)-protein complex comprising washingthe complex with an alkanediol.

In yet further embodiments, the present invention comprises processesfor increasing the retention time of a protein on a resin comprising thestep of washing the resin with an alkanediol. In an example of such anembodiment, after a wash with a hexanediol, such as 1,2-hexanediol, toremove the LPS, increasing the salt concentration on the resin fails toelute the protein. Such embodiments are useful for high saltenvironments and where one may wish to alter the selectivity of theresin for proteins.

Accordingly, embodiments of the present invention further comprisemethods of separating LPS from a protein-LPS complex in a high salt,conductivity, environment comprising the steps of:

immobilizing the complex to an ion exchange resin;

washing the resin with 1,2-hexanediol whereby at least a portion of theLPS is separated from the complex; and,

eluting at least a portion of the protein from the resin by modifyingthe pH. In preferred embodiments of this type, the concentration ofalkanediol is greater than about 5%. However, any concentration may beused.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and the appended Claims are intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth whether now existing or afterarising. Further, while embodiments of the invention have been describedwith specific dimensional characteristics and/or measurements, it willbe understood that the embodiments are capable of different dimensionalcharacteristics and/or measurements without departing from theprinciples of the invention and the appended Claims are intended tocover such differences. Furthermore, all patents mentioned herein areherby incorporated by reference.

For a further understanding of various embodiments of the presentinvention, reference should be had to the following examples:

EXAMPLES AND EXPERIMENTS

Materials

Bovine albumin (BSA), bovine holo-transferrin, lactoferrin from bovinemilk, lysozyme from chicken egg whites, lipopolysaccharides fromEscherichia coli serotype O55:B5, and BSTFA were purchased from SigmaChemical Co. (St. Louis, Mont.). Acetic acid, Tris (base), sodiumhydroxide (NaOH), hydrochloric acid, sodium chloride (NaCl), ethanol,isopropanol, sodium dodecyl sulfate (SDS), and sodium phosphate dibasic7-hydrate were purchased from J.T. Baker Chemical Co. (Phillipsburg,N.J.). 1,6-Hexanediol was from BASF Co. (Mount Olive, N.J.).1,2-Hexanediol, 1,2-butanediol, and Zwittergent 3-14 (Zw 3-14) werepurchased from Fluka (Milwaukee, Wis.). 1,4-Butanediol and ethyleneglycol were purchased from Aldrich (Milwaukee, Wis.). Phosphate bufferedsaline (PBS), 10×, was purchased from Bio-Rad Laboratories, Inc.(Hercules, Calif.). Escherichia coli BODIPY® FL conjugatelipopolysaccharide, serotype O55:B5, (BODIPY-LPS) and EnzChek LysozymeAssay Kit were purchased from Molecular Probes, Inc. (Eugene, Oreg.).Pyrosol, Limulus Amebocyte Lysate (LAL) Pyrotell-T, LAL reagent water(LRW), and control standard endotoxin from E. coli O113:H10 (CSE), wereobtained from Associates of Cape Cod, Inc. (Falmouth, Mass.). SPSepharose fast flow (SPFF) resin, Q Sepharose fast flow (QFF) resin, andHR 10/10 columns were from Amersham Biosciences (Piscataway, N.J.).Clear polystyrene 96-well microtiter plates were from Associates of CapeCod, Inc. (Falmouth, Mass.) and black 96-well microtiter plates fromNUNC (Rochester, N.Y.). β-Hydroxytetradecanoic acid,β-hydroxytridecanoic acid, β-hydroxyundecanoic acid,β-hydroxytridecanoate, β-hydroxytetradecanoate, and β-hydroxyundecanoatewere purchased from Matreya, Inc. (Pleasant Gap, Pa.). Heptane waspurchased from Spectrum (New Brunswick, N.J.).

Methods

Viscosity Measurements

A Brookfield Model 1 LVT Viscometer equipped with a ULA-Y adapter(Middleboro, Mass.) was used for viscosity determinations. Allmeasurements were performed at 20° C.

LPS-Protein Complex Formation

Protein stock solutions were prepared in the column equilibrationbuffers to a final concentration of 10 to 11 mg/ml. LPS O55:B5 stocksolution was prepared in Milli-Q water and BODIPY-LPS stock solution wasprepared in PBS or column equilibration buffers to a final concentrationof 1 mg/ml. This is approximately 100 μM final concentration based on amolecular weight of O55:B5 LPS of 10,000 Da.

The LPS-protein complexes were formed by adding to a polypropylene tube1 part LPS solution to 9 parts protein solution, v/v. The tube wasvortexed, wrapped in aluminum foil, and then incubated at roomtemperature for 16 to 72 hours for BSA and transferrin or incubated at37° C. for at least 4 hours for lactoferrin.

LAL and BODIPY Analysis

A SpectraMax Gemini XS microplate spectrofluorometer from MolecularDevices Co. (Sunnyvale, Calif.) was used for the BODIPY-LPS fluorescentmicroplate assay and a SpectraMax 190 microplate spectrophotometer(Molecular Devices) for the LAL kinetic turbidimetric assay (KTA).Reference materials were analyzed in triplicate and samples either induplicate or triplicate. The results were plotted and analyzed usingSOFTmax PRO software version 3.1.1 (Molecular Devices).

A. BODIPY-LPS Assay

Three fold serial dilutions of the BODIPY-LPS stock solution wereprepared from 0.81 to 0.03 μM. The analytical procedure of theBODIPY-LPS assay was a modification of the assay used by Yu and Wright(1996) as follows. To each well of a black microtiter plate 20 μl of 15%SDS, prepared in Milli-Q water, followed by 180 μl sample or standardwere added. The plate was shaken for 10 seconds at 37° C. and readimmediately in fluorescence mode. The optimal excitation (490 nm),emission (525 μm), and cutoff wavelengths (515 nm) were determined forthe BODIPY-LPS. The assay for BODIPY-LPS demonstrated a linear rangefrom 0.03 to 0.81 μM (54 to 1458 ng) with a limit of detection of/LOQvalues less than 0.01 μM (18 ng) and a limit of quantification of 0.03μM (54 ng).

B. LAL Kinetic Turbidimetric Assay (KTA)

Samples were adjusted to a pH between 6 and 8 with Pyrosol, if needed.CSE and Pyrotell-T were reconstituted with LRW. The linear curve of CSEwas from 0.03 to 1.00 EU/ml. The analytical procedure of LAL KTA was asfollows. To each well of a clear polystyrene microtiter plate 100 μl ofsample or standard and 100 μl of Pyrotell-T were added. For spikedsamples, 5 μl of 2.00 EU/ml CSE was added to obtain 0.10 EU/ml CSElevel. The plate was shaken for 10 seconds and data collected, everyminute, in the kinetic mode at 405 nm for 1 hour at 37° C.

C. LPS Analysis by Gas Chromatography

Quantitative analysis for the relative level of LPS present in samplesby gas chromatography mass spec (GCMS) was based on the method byMielniczul et al. (1992) and utilized a 6890 gas chromatograph with a5973 mass selective detector from Agilent (Foster City, Calif.). Thecolumn used was a DB-5MS column (30 cm×0.25 mm i.d.×0.25 μm filmthickness) from J&W Scientific (Foster City, Calif.). The linear curvefor surrogate and target compounds ranged from 1.6 to 100 pg/μl and theinternal standard was kept constant at 50 pg/μl. Briefly, the method ofsample preparation and GCMS analysis were as follows. Samples and aknown amount of surrogate, β-hydroxytridecanoic acid, were added to 5 mlglass reaction vials. Aqueous samples were hydrolyzed in 6 N HCl at 90to 100° C. overnight to liberate β-hydroxytetradecanoic acid from LPS.The fatty acids were extracted twice from the hydrolysate with heptaneand then dried under nitrogen. The fatty acids were methylated byincubation at 80 to 90° C. in 3 N methanolic HCl for 30 minutes. Waterwas added to quench the reaction and then the methyl esters extractedtwice with heptane. The methyl esters were then dried under nitrogen.The methyl esters were derivatized by adding BSTFA/pyridine (2:1, v/v)and incubating at 80 to 90° C. for 15 to 20 minutes before undergoingthe final drying step under nitrogen. Samples were reconstituted with a50 pg/μl internal standard, methyl-3-trimethylsilyl-undecananoic acid,prepared in heptane. Standards and samples were injected in splitlessmode and at 1 μl injection volume. Initial oven temperature was held at90° C. for 4 minutes and then ramped at 20° C. per minute to 250° C.followed by a 10° C. per minute ramp to 300° C. The mass spectrometerwas set for an EM offset voltage of 500 and the solvent delay at 5.2minutes. Selective ion monitoring was used to monitormethyl-3-TMS-undecanoate at ions 175 and 273, methyl-3-TMS-tridecanoateat 11.0 minutes and ions 175 and 301, and methyl-3-TMS-tetradecanoate at11.7 minutes and ions 175 and 315. Chromatograms were reported usingChemstation for MSD Productivity software.

Lysozyme Assay

Lysozyme activity was determined using the EnzChek Lysozyme Assay Kitaccording to the manufacturers instructions.

Chromatography

All chromatography was performed on ÄKTA explorer 100 FPLC systems(Amersham Biosciences) equipped with a gradient pump (P-900), a 500 μlor 2000 μl injection loop, a variable wavelength detector (UV-900), anda pH and conductivity monitor (pH/C-900). All chromatographicexperiments were performed at a flow rate of 200 to 300 cm/hr at ambienttemperature. During the alkanediol washes the flow rate was dropped to150 to 200 cm/hr to minimize the increase in system back pressure due tothe increased viscosity of the alkanediol solutions. Chromatograms werereported using Unicorn software version 3.21 or 4.0. The resins werepacked in 1 cm diameter columns to bed heights of 7 to 11 cm. The ÄKTAsand columns were sanitized either with 0.5 N NaOH for 60 to 120 minutesor 0.1 N NaOH for greater than 16 hours before each chromatographic run.The column and ÄKTAs were then rinsed with Milli-Q water just prior tosystem equilibration with the appropriate buffers.

Alkanediols, ethanol, and isopropanol were prepared as v/v solutionswith the same chemical compositions and pH as the equilibration buffers,while 1,6-hexanediol and Zwittergent 3-14 were prepared as w/vsolutions.

D. Cation Exchange Chromatography

For transferrin, a SP Sepharose Fast Flow column was charged with 100 mMAcetate, 1 M NaCl, pH 5, and equilibrated with 100 mM Acetate, pH 5.After loading, the resin was washed with the equilibration buffer andthen eluted with 50 mM sodium phosphate, 1 M NaCl, pH 7.5. When anorganic or detergent wash was performed, it was applied after theinitial wash step and was for 6 CV unless otherwise stated. This washwas followed by a second wash with equilibration buffer to remove theorganic or detergent prior to elution.

The chromatography for BSA was identical to that of transferrin exceptthat the pH of all chromatography buffers was 4.5. When 1,2-hexanediolwas used as the washing agent, the eluent was changed to 50 mM sodiumphosphate, 1 M NaCl, pH 7.5.

Lactoferrin chromatography was similar to transferrin except that theresin was charged with 1 M sodium chloride, 20 mM sodium phosphate, pH7.5, equilibrated in 20 mM sodium phosphate, pH 7.5, and eluted with 1 Msodium chloride, 20 mM sodium phosphate, pH 7.5.

The chromatography for lysozyme was similar to that for transferrinexcept that the resin was charged with 1 M NaCl, 20 mM Tris, pH 8.0,equilibrated in 50 mM Tris, pH 8.0 and eluted with 1 M NaCl, 20 mM Tris,pH 8.0.

E. Anion Exchange Chromatography

For BSA, a Q Sepharose Fast Flow column was charged with 50 mM Tris, 1 MNaCl, pH 8.0 and equilibrated with 50 mM Tris, pH 8.0. After loading,the resin was washed with equilibration buffer. BSA was eluted with 25mM Acetate, pH 4.5, and LPS with 25 mM Acetate, 1 M NaCl, pH 4.5. Whenan alkanediol wash was performed, it was inserted after the initial washstep and was for 6 CV. This wash was followed by a second wash withequilibration buffer to remove the alkanediol.

Results and Discussion

Table I summarizes the molecular weights and isoelectric points of theproteins used in this study. BSA, transferrin, and lactoferrin have allbeen shown to bind LPS (Dzarski, 1994; Berger and Beger, 1987; andAppelmelk et al., 1994). Lysozyme was used to assess the effects of thewashing agents on enzyme activity. TABLE I Molecular Weights andIsoelectric Points of Proteins Used in this Study Protein MolecularWeight pI Albumin, bovine fatty acid free low 66400 5.56 endotoxinTransferrin, bovine holo 75800 6.5 Lactoferrin, bovine 75200 8.52Lysozyme, chicken egg white 14300 9.65

Table II summarizes some of the physical properties of the alkanediolsused in this study. Also included are ethanol and isopropanol, whichhave been used for LPS removal in other processes (Franken et al.,2000), for comparison. TABLE II Physical Properties of Alkanediols Usedin this Study ¹Boiling ¹Melting ¹Flash ²Explosion limits, air²Autoignition Compound Point (° C.) Point (° C.) Point (° C.) LowerUpper (° C.) 1,2-hexanediol 223 NA 122 NA NA 390 1,6-hexanediol 250 45147 6.6%   16% 319 1,5-pentanediol 242 −16 129 1.4% 13.2% NA1,2-butanediol 194 −50 93 2.4% 13.5% 390 1,4-butanediol 230 16 1211.95%  18.3% 420 1,3-propanediol 214 −27 131 NA NA 400 1,2-ethanediol195 −13 111 3.2% 15.3% 400 Ethanol 78.3 −114.1 12 3.3%   19% 363Isopropanol 82.4 −88.5 12 2.5%   12% 460¹data obtained from CabridgeSoft Corp. at Chemfinder.com²data from Material Data Safety SheetsNA, not availableSeparation of LPS-Protein Complexes by Organics and Detergent

SP Sepharose Fast Flow Chromatography of LPS and LPS-Protein Complexes

The LPS elution profiles of LPS by itself and LPS-BSA complexes on SPSepharose Fast Flow resin were determined by LAL-KTA analysis ofselected column fractions (Table III). When LPS was chromatographed byitself the LPS was detected primarily in the wash-unbound fraction asexpected. Chromatography of the LPS-BSA complexes resulted in themajority of the LPS being detected in the BSA eluate fraction confirmingthe LPS binding property of BSA (Dziarski, 1994) and demonstrating thatthe BSA-LPS complexes are stable under cation exchange chromatographyconditions employed. TABLE III SPFF Chromatography Elution Profiles ofLPS and LPS-BSA LPS and LPS-BSA complex were chromatographed on SPSepharose Fast Flow. Column fractions were analyzed for LPS by the LALKTA as described in the methods. BODIPY-LPS (% Recovery) Sample LoadWash-unbound Elution BODIPY-LPS 100 82 5 BODIPY-LPS-BSA Complex 100 3 90

The LAL KTA is a laborious and costly assay to use to determine thedistribution of LPS in the column fractions. A fluorescent based assayfor LPS was developed to monitor the column fractions. This assay usedfluorescently tagged LPS, BODIPY-LPS, in place of the non-labeled LPS,which allows for the quick analysis of the column eluates byfluorescence spectroscopy. The fluorescence of the BODIPY maker in theBODIPY-LPS conjugate has been shown to be quenched when the LPS iscomplexed with itself or protein. Addition of SDS to the sample disruptsthe LPS-LPS or LPS-protein complexes and results in an increase influorescence (Yu and Wright, 1996). The assay was developed as amicrotiter plate based assay that allowed for the quick and quantitativeanalysis of BODIPY-LPS in the chromatography fractions.

To determine if the BODIPY marker interfered with BSA-LPS complexformation or behaved differently during cation exchange chromatographythe preceding analysis was repeated using BODIPY-LPS andBODIPY-LPS-protein complexes. The elution profiles of BODIPY-LPS andBODIPY-LPS-protein complexes were similar (Table IV) to the elutionprofiles of LPS and LPS-BSA above. This demonstrates that the BODIPYmarker does not interfere with the ability of BSA or transferrin to bindLPS and that the BODIPY group does not alter the chromatographic profileof the LPS. FIG. 1 shows typical SP Sepharose Fast Flow profiles forBODIPY-LPS (A) and the BODIPY-LPS-transferrin complex (B). TABLE IV SPFFChromatography Elution Profiles of BODIPY-LPS and BODIPY-LPS-ProteinBODIPY-LPS and BODIPY-LPS-protein complex were chromatographed on SPSepharose Fast Flow. Column fractions were analyzed for BODIPY-LPS bythe BODIPY assay as described in the methods. BODIPY-LPS (% Recovery)Sample Load Wash-unbound Elution BODIPY-LPS 100 82 5 BODIPY-LPS-BSAComplex 100 2 90 BODIPY-LPS-Transferrin 100 6 74 ComplexReduction of LPS from LPS-Protein Complexes by Alkanediols During SPSepharose Fast Flow Chromatography

Initial experiments examined the capability of a 50% 1,6-hexanediol washstep to reduce the amount of BODIPY-LPS complexed with BSA during cationexchange chromatography. A 3 column volume (CV) wash with 1,6-hexanediollowered the amount of BODIPY-LPS complexed with BSA by about 21%.Increasing the length of the 1,6-hexanediol wash step from 3 CV to 6 CVimproved the removal of BODIPY-LPS from the BSA complex to about 49%.While an additional 3 CV increase in the 1,6-hexanediol wash step to 9CV provided improvement (51%) in BODIPY-LPS removal. All additionalexperiments were carried out with a 6 CV alkanediol wash step.

The effectiveness of a series of alkanediols to remove LPS from proteinswhile the proteins were bound to ionic solid supports were compared tothose of ethanol, isopropanol, and Zwittergent 3-14, which had beenshown to be effective in removing LPS from a LPS binding protein. A SPSepharose Fast Flow column was loaded with BODIPY-LPS-transferrincomplex. The resin was washed with six column volumes of a 50%alkanediol solution and then eluted. Fractions were collected andassayed for BODIPY-LPS (FIG. 2). As the chain length of the alkanediolwas increased from four to six carbons, the fluorescence of thealkanediol wash fractions increased while the fluorescence of the eluatefractions decreased. This demonstrated that alkanediols removedBODIPY-LPS from the transferrin complex. FIGS. 3 and 4 illustrate theeffects of alkanediol structure on BODIPY-LPS removal from transferrinand BSA, respectively. BODIPY-LPS removal efficiency increased withincreasing alkanediol chain length and the 1,2-alkanediol isomers weremore effective than the terminal alkanediols at removing the BODIPY-LPS.1,2-hexanediol was the most efficient compound tested and out performedthe detergent and alcohols. 1,2-butanediol and 1,6-hexanediol as well as50% isopropanol and 75% ethanol reduced the BODIPY-LPS associated withtransferrin to similar levels. The removal of BODIPY-LPS by thealkanediols was similar for both the transferrin and BSA complexes.

Reference to FIG. 2 illustrates BODIPY-LPS Elution Profiles ofBODIPY-LPS-Transferrin Complexes on SP Sepharose Fast Flow inConjunction with Alkanediol Washes.

FIG. 3 illustrates the Reduction of BODIPY-LPS from BODIPY-LPSTransferrin Complexes in SP Sepharose Fast Flow Eluates by Alkanediols.Chromatography was as described in FIG. 2. Zero percent reductioncorresponds to a control run without an alkanediol wash. 1,1,2-hexanediol; 2, 1% Zwittergent 3-14; 3, 1,6-hexanediol; 4, ethyleneglycol; and 5, 1,4-butanediol. BODIPY-LPS-transferrin complexes weregenerated, loaded onto a SP Sepharose Fast Flow column and the resinwashed with 50% solutions of 1,4-butanediol, 1,6-hexanediol, or1,2-hexanediol. Following a wash to remove the alkanediol, transferrinwas eluted as described in the methods. Sanitization between runs waswith 0.5 N NaOH.

FIG. 4 illustrates The reduction of BODIPY-LPS from BODIPY-LPS-BSAcomplexes in SP Sepharose Fast Flow eluates by alkanediols.Chromatography was as described in FIG. 2. Zero percent reductioncorresponds to a control run without an alkanediol wash. 1,1,2-hexanediol; 2, 1% Zwittergent 3-14; 3, 1,2-butanediol; 4,1,6-hexanediol; 5, 50% isopropanol; 6, 75% ethanol; 7, 1,4-butanediol;and 8, ethylene glycol.

It was noted during the experiments on transferrin utilizing1,2-hexanediol in the wash that when the pH of the elution buffer wasmaintained at a pH of 5, including 1 M NaCl, that the transferrin wasnot eluted from the resin. Increased retention of transferrin wasobserved down to a 1,2-hexanediol concentration of 10%. At a1,2-hexanediol concentration of 5%, transferrin retention was notaltered. The alteration in retention times has been observed for otherproteins during ion exchange chromatography in the presence ofpolyethylene glycol and other neutral polymers (Milby et al., 1989 andGagnon et al., 1996). The changes in protein retention times weredependent on polyethylene glycol size and concentration and the proteinitself. Whereas, ethylene glycol up to a concentration of 40% did noteffect protein retention times (Tauc et al., 1998). It is worth while tonote that in these instances the compound under investigation wereincluded in all the chromatography buffers. For the 1,2-hexanediol, itwas only included in a wash buffer that was then removed by anadditional wash step prior to elution of the protein.

Since 1,2-hexanediol was the most effective compound tested for removingBODIPY-LPS from both transferrin and BSA, the concentration dependenceof the 1,2-hexanediol wash needed to affect this removal wasinvestigated. The reduction of BODIPY-LPS in the SP Sepharose Fast FlowBSA eluate fraction was determined after 1,2-hexanediol washescontaining 5%, 20%, and 50% 1,2-hexanediol. The 5% 1,2-hexanediol washresulted in about a 55% decrease in the BSA associated BODIPY-LPS whilethe reduction of BODIPY-LPS by the 20% and 50% 1,2-hexanediol washeswere comparable at approximately 96%.

In addition to transferrin and BSA, the removal of BODIPY-LPS fromlactoferrin complexes by 1,6-hexanediol was also examined by thefluorescent BODIPY assay. The results of the analysis were thenconfirmed by analysis of the samples for the LPS marker compound3-OH-14:0 fatty acids by GC-MS. Table V summarizes the data and againdemonstrates the ability of an alkanediol, in this case 1,6-hexanediol,wash during the chromatography to reduce the levels of BODIPY-LPS in thelactoferrin eluate. Approximately and 87% reduction of BODIPY-LPS wasobserved by the BODIPY fluorescence assay and a 91% reduction in the LPSmarker by the GC-MS assay. TABLE V SPFF Chromatography Elution Profilesof BODIPY-LPS and BODIPY-LPS-Lactoferrin ComplexesBODIPY-LPS-lactoferrin complexes were generated, loaded onto a SPSepharose Fast Flow column. For runs that included a 50% solution of1,6-hexanediol, a wash to remove the 1,6-hexanediol was included priorto elution of lactoferrin as described in the methods. Column fractionswere assayed for BODIPY and 3-OH-14:0 fatty acids as described in themethods. BODIPY-LPS (% Recovery) GCMS assay/Fluorescence BODIPY assaySample Load Wash-unbound Diol wash Eluate BODIPY-LPS 100 (GCMS) 94 ¹N/A 5 (no diol wash) 100 (fluor) 89 N/A 7 BODIPY-LPS- 100 62 N/A 48Lactoferrin (no diol wash) 100 60 N/A 46 BODIPY-LPS- 100 63 ²ND  4Lactoferrin (+ diol wash) 100 66 26 6¹N/A—Not applicable.²ND—Could not be determined by GCMS.

During the chromatographic runs, a rise in the system back pressure wasnoted when the alkanediol washes were applied. The viscosity of eachorganic solutions and the Zwittergent solution, prepared in 100 mMAcetate, pH 4.5, were measured (FIG. 5). The viscosity of thealkanediols increased with carbon chain length while the viscosity ofthe 1,2-alkanediol isomers were slightly less than the terminalalkanediol isomers. The increased viscosity of the alkanediol solutionsmay present some difficulties in scale-up. Column flow rates may have tobe adjusted to maintain suitable system pressure for the equipment inuse. Being able to use lower concentrations of the alkanediols to removeLPS from the protein-LPS complexes would partially alleviate thisproblem. For example, 20% and 50% 1,2-hexanediol washes effectivelyreduce the BODIPY-LPS to approximately the same levels for BSA complexesas indicated above. The viscosity of 20% 1,2-hexanediol is about onethird that of 50% hexanediol, 2.6 Cp compared to 7.5 Cp.

Reduction of LPS from LPS-Protein Complexes by Alkanediols during QSepharose Fast Flow Chromatography

Removal of LPS from LPS-protein complexes is more complex on anionexchange resins, especially for basic proteins. During cation exchangechromatography, the LPS, being negatively charged, is not attracted tothe functional group of the resin and is washed out of the column duringthe alkanediol wash while the protein remains bound under the washconditions. During anion exchange chromatography, the LPS and theprotein both are retained by the resin's functional groups. Thereforethe complexes need to be disrupted and differential elution of theprotein and LPS must occur.

The ability of 1,6-hexanediol and 1,2-hexanediol to reduce theBODIPY-LPS levels of BSA-LPS complexes during anion exchangechromatography on Q Sepharose Fast Flow resin was investigated. The twoisomers, 1,2- and 1,6-, of hexanediol were chosen since these were themost effective compounds at reducing the levels of BODIPY-LPS fromprotein complexes during cation exchange chromatography.

The elution of the BSA from the anion exchange resin after theapplication of a 1,2-hexanediol wash was altered as was observed fortransferrin on the cation exchange resin. Increasing the saltconcentration of the elution buffer to affect the elution of BSA afterthe 1,2-hexanediol wash resulted in the coelution of free BODIPY-LPSwith the BSA. An alternative elution scheme was chosen based on pH.After the 1,2-hexanediol wash was complete and a wash out of the1,2hexanediol had occurred, the BSA was eluted at pH 4.5. This wasfollowed by a strip of the resin, which brought off the remainingBODIPY-LPS, with a pH 4.5 buffer containing 1 M NaCl. Table VIsummarizes the effect of 1,2-hexanediol on the removal of BODIPY-LPSfrom BODIPY-LPS-BSA complexes. Inclusion of the 1,2-hexanediol washreduced the BODIPY-LPS of the BSA eluate by about 29% with an apparentelution of the displaced BODIPY-LPS to the 1,2-hexanediol washfractions. TABLE VI The QFF Elution Profiles of BODIPY-LPS andBODIPY-LPS-BSA Complexes. BODIPY-LPS and BODIPY-LPS-BSA complex werechromatographed on Q Sepharose Fast Flow. Column fractions were analyzedfor BODIPY-LPS by the BODIPY assay as described in the methods.BODIPY-LPS (% Recovery) Wash- Sample Load unbound Diol wash Elute StripBODIPY-LPS 100 1 ¹NA  0 42 (no diol wash) BODIPY-LPS-BSA 100 7 NA 17 52(no diol wash) BODIPY-LPS-BSA 100 2 12 5 45 (+ diol wash)¹NA—Not applicable.Alkanediol Effect on Enzymatic Activity

Lysozyme was used to determine the effect of the washing agents onenzymatic activity and thereby, indirectly the denaturing effects of thewashing agents during SP Sepharose Fast Flow chromatography. Lysozymewas chromatographed with and without a 6 CV %50 1,6-hexanediol wash or1,2-hexanediol wash and the column loads and eluates assayed forlysozyme activity using a fluorescence microplate lysozyme activityassay. The hexanediol washes had no detrimental effects on lysozymeactivity. 75% ethanol, 50% isopropanol, and 1% Zwittergent 3-14 washesalso had no effect on lysozyme activity.

REFERENCES

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1. A process for removing an endotoxin from recombinantly producedproteins comprising a Lipopolysaccharide (LPS)-protein complexcomprising the steps of: immobilizing the complex to an chromatographicresin; washing the resin with an alkanediol whereby at least a portionof the LPS is separated from the complex; and, eluting at least aportion of the protein from the resin.
 2. The process of claim 1 whereinthe alkandiol is selected from the group consisting of 1,5-pentanediol,1,6-hexanediol, 1,2-hexanediol, 1,2-butanediol, 1,4-butanediol, and1,7-heptanediol.
 3. The process of claim 1 wherein the resin is selectedfrom the group consisting of a cation exchange resin and an anionexchange resin.
 4. The process of claim 1 wherein the protein isselected from the group consisting of bovine albumin (BSA), bovineholo-transferrin, lactoferrin, lysozyme, and heat shock proteins.
 5. Theprocess of claim 1 wherein the protein is affixed to the resin.
 6. Theprocess of claim 1 wherein the LPS is affixed to the resin.
 7. Theprocess of claim 1 wherein greater than about 95% of the protein elutedfrom the resin is free of LPS or other endotoxin.
 8. The process ofclaim 7 wherein a change in pH or conductivity is used to elute theprotein from the resin.
 9. The process of claim 1 wherein the protein isproduced from a bacterial expression system.
 10. The process of claim 9wherein the bacterial expression system is selected from the groupconsisting of an E. coli expression system, a Caulobacter crescentexpression system, and Proteus mirabilis expression system.
 11. Theprocess of claim 1 wherein the resin is in a high salt environment. 12.The process of claim 11 wherein the alkanediol is 1,2-hexanediol. 13.The process of claim 12 wherein the protein is eluted by changing thepH.
 14. A process for separating an endotoxin from a protein comprisingthe steps of affixing the protein to a resin and washing the resin withan alkanediol whereby at least a portion of the endotoxin is separated.15. The process of claim 14 wherein the endotoxin is an impurity createdfrom the production of the protein.
 16. The process of claim 14 whereingreater than 99.9% of the enndotoxin is separated from the protein. 17.A process for removing an endotoxin from recombinantly produced proteinscomprising a Lipopolysaccharide (LPS)-protein complex comprising thesteps of: affixing the protein of the complex to a cation exchangechromatographic resin; washing the resin with an alkanediol whereby atleast a portion of the LPS is separated from the complex; and, elutingat least a portion of the protein from the resin by either changing thepH or the conductivity.
 18. The process of claim 17 wherein the LPS ofthe complex is affixed to an anion exchange chromatographic resin.